A multiple antigen peptide from the repetitive sequence of the Plasmodium malariae circumsporozoite protein induces a specific antibody response in mice of various H-2 haplotypes

The major repetitive epitopes of the surface circumsporozoite (CS) protein of malaria sporozoites represent candidates for the development of subunit vaccines against malaria. However, previous experimental work has shown that repetitive peptides from the CS proteins of Plasmodium falciparum, P. vivax, P. yoelii and P. berghei are immunogenic only in mice with the H-2b or H-2k haplotype. This led to the conclusion that strong T helper epitopes from the non-repetitive CS sequences were required in the design of sporozoite vaccines. In the present study, we investigated the immunogenicity in mice of a octa-branched multiple antigen peptide (MAP) containing repeats of the CS protein of the human malaria parasite, P. malariae, [MAP8(NAAG)6], and found that mice with an H-2b, H-2d, H-2k, H-2f, H-2q, and H-2s haplotype produced anti-peptide antibodies after immunization and that only H-2r mice were nonresponsive. This antibody response, not induced in athymic H-2b nu/nu mice, was directed against the (NAAG) sequence, but not against the lysine core of the MAP construct. Finally, when covalently linked to a synthetic polymer of the repetitive (NANP) sequence of the P. falciparum CS protein, [MAP8(NAAG)6] behaved as a carrier molecule for the production of anti-(NANP)n antibodies in H-2d and H-2k mice, genetically nonresponder to the (NANP)n sequence. Should this wide immunogenicity of the P. malariae CS (NAAG) repetitive sequence also apply to humans, it might be considered for the design of multivalent subunit malaria vaccines.     

Immunogenicity of a non-repetitive sequence of Plasmodium falciparum circumsporozoite protein in man and mice

In the present work, the hypothesis that individuals naturally exposed to Plasmodium falciparum malaria infection in endemic areas produce antibodies directed against non-repetitive epitopes of the circumsporozoite protein was investigated. Using a synthetic peptide reproducing the non-repetitive group-conserved region I sequence, we have shown that specific anti-region I antibodies are detectable in sera from endemic countries. Of these sera, 87% also had antibodies against the immunodominant repetitive epitope (Asn-Ala-Asn-Pro, NANP) of P. falciparum. In order to study the immunogenicity of this non-repetitive epitope, a synthetic peptide consisting of both region I and three (NANP) repeats [RI-(NANP)3] was used to immunize inbred strains of mice. H-2b mice produced antibodies against both the repetitive and the non-repetitive epitope. These antibodies were specific for each epitope, recognized P. falciparum sporozoites in immunofluorescence, and inhibited sporozoite penetration into human liver cells in vitro. Non-H-2b mice were completely unresponsive. Lymph node cells from H-2b mice immunized with RI-(NANP)3 peptide proliferated in the presence of RI-(NANP)3 and of (NANP)4 peptide, but never in the presence of RI peptide alone. These findings demonstrate that in the configuration used (i) the non-repetitive epitope region I does not carry T-helper epitopes; (ii) the (NANP) repetitive epitope may act as a carrier for the immune response to region I in mice; and (iii) therefore, immune response to region I in man probably depends on the recognition of T-cell epitopes similar to those involved in the anti-NANP response: i.e. such a T epitope may be NANP itself in responding individuals or another, not yet recognized, sporozoite T-cell epitope.     

Primary and Secondary Responses to (NANP) Peptides by Plasmodium falciparum Sporozoites in Various Strains of Mice

The repetitive epitope (Asn-Ala-Asn-Pro = NANP) of the Plasmodium falciparum circumsporozoite protein is considered as the basis for the development of a recombinant or synthetic subunit vaccine against malaria. Vaccines consisting of (NANP)n molecules coupled to carrier proteins have already been tested in trials in human volunteers with partial success. In this paper we show that C57BL/6 mice, genetically responsive to carrier-free (NANP)n molecules, exhibit a secondary antibody response to (NANP) if they are primed with carrier-free (NANP)40 synthetic peptide, and then challenged with P. falciparum sporozoites. However, such a sporozoite-mediated boosting effect is not observed if C57BL/6 and BALB/c mice were previously primed with (NANP)40 peptide conjugated to carrier proteins. The genetic restriction of the murine antibody response to (NANP)n is overcome when mice bearing seven different H-2 haplotypes are immunized with entire P. falciparum sporozoites. These results may have implications for the understanding of natural or induced anti-sporozoite immunity, and show that the use of T-cell epitopes from the plasmodial antigenic repertoire would be very likely to represent an efficient approach for the development of a subunit malaria vaccine.     

Characterization of murine monoclonal antibodies against a repetitive synthetic peptide from the circumsporozoite protein of the human malaria parasite, Plasmodium falciparum

Monoclonal antibodies (mAbs) were raised in mice against the synthetic peptide (NANP)40, consisting of 40 (NANP) repeats of the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium falciparum, and characterized. (i) Five of these mAbs recognized the P. falciparum CS protein in western blot experiments and in immunofluorescence assays using different preparations of sporozoites. The remaining two mAbs (CT3.2 and CT3.3, both IgG1) gave negative results by both techniques. (ii) When the anti-(NANP)40 peptide mAbs were functionally tested in vitro to assess their ability to inhibit the attachment and penetration of the parasites into cultured human liver cells, six of them exhibited inhibitory activities ranging between 66 and 90%. CT3.2 mAbs, also, inhibited sporozoite attachment and penetration, despite the negative results by immunofluorescence and western blot experiments. However, when immunofluorescence was repeated in the presence of calcium, CT3.2 did reveal a positive recognition of P. falciparum sporozoites, suggesting that this mAb could recognize the (NANP) sequence when calcium was bound to the repetitive peptide. (iii) Furthermore, the binding of an anti-(NANP)40 IgM mAb (CT1) to the solid-phase peptide was not inhibited by preincubation of the peptide with a mAb against the P. falciparum CS protein. (iv) Finally, one anti-(NANP)40 IgG1 mAb (CT3.1) was unable to bind to the shorter (NANP)3 peptide, although it recognized the (NANP)40 peptide and the P. falciparum CS protein. The results presented here suggest that heterogeneous antibody populations are produced upon immunization of mice with (NANP)40 synthetic peptide and that epitopes different from those simply related to the linear (NANP) amino acid sequence are likely to be present in long (NANP)n constructs as well as in the repetitive domain of the P. falciparum CS protein.     

Detection of human antibodies against Plasmodium falciparum sporozoites using synthetic peptides.

A large peptide consisting of about 40 (Asn-Ala-Asn-Pro) repeats of Plasmodium falciparum circumsporozoite protein, (NANP)40, was synthesized. It was recognized specifically by monoclonal antibodies produced against P. falciparum sporozoites. Moreover, this peptide strongly inhibited the binding of such monoclonal antibodies to antigens present in a sporozoite extract. The (NANP)40 peptide was employed without any carrier to develop an enzyme-linked immunosorbent assay to detect sporozoite-specific serum antibodies arising after natural malaria infections. Antibodies were detected in a high percentage (43.1%) of European patients suffering from acute P. falciparum malaria and in Africans living in an area of Gabon endemic for malaria. In the latter group, the frequency of antisporozoite antibodies increased with age, reaching 65.9% in individuals more than 40 years old. There was a significant correlation between the results obtained with an immunofluorescence assay with glutaraldehyde-fixed sporozoites and those obtained by enzyme-linked immunosorbent assay with (NANP)40. Therefore, such synthetic peptides representing the repetitive epitope of P. falciparum circumsporozoite protein can be used for the detection of antisporozoite antibodies and for the epidemiological studies required to obtain base-line data concerning the immune status of individuals before their participation in a sporozoite vaccine trial.     

Reduced antibody response to the repetitive sequence of the Plasmodium falciparum circumsporozoite protein in mice infected with Plasmodium yoelii blood forms

The immunogenicity of the carrier-free synthetic peptide, (NANP)₄₀, from the repetitive region of the Plasmodium falciparum circumsporozoite (CS) protein was investigated in genetically responder mice (C57BL/6, H-2^b) acutely infected with blood forms of the non-lethal murine malaria parasite, P. yoelii. As compared to non-infected mice, P. yoelii-infected C57BL/6 mice produced significantly lower titers of anti-(NANP)₄₀ IgG antibodies. This decrease in the anti-(NANP)₄₀ antibody response peaked with the peak of parasitemia, and involved all the IgG subclasses. Interestingly, this P. yoelii-mediated effect was evident both on the development of the antibody response to the (NANP)₄₀ peptide, and on an already established anti-(NANP)₄₀ antibody titer, as seen in mice immunized with the peptide 1 month before the infection. Since (NANP)_n-based constructs are strongly envisaged as potential vaccines against falciparum malaria, these results might be important in the evaluation of the efficacy of these vaccine candidates, when they will be used in individuals living in endemic areas.     

Immune responses to defined epitopes of the circumsporozoite protein of the murine malaria parasite, Plasmodium yoelii.

We have investigated the immunogenicity of defined sequences of the circumsporozoite (CS) protein of the murine malaria parasite, Plasmodium yoelii. A 21-ner synthetic peptide from the nonrepetitive region of the CS protein (position 59-79, referred to as Py1) induced T cell proliferative responses in H-2d and, to a lesser extent, in H-2b mice. Conversely, a synthetic peptide (referred to as Py4) consisting of four (QGPGAP) repeats of the P. yoelii CS protein, induced an antibody response only in H-2b mice. No antibody response was observed when the Py3 peptide, consisting of three (QGPGAP) repeats, was used as an immunogen. When cross-linked to the Py4 repetitive peptide, the Py1 sequence behaved as a T helper epitope allowing the production of anti-Py4 antibodies in H-2d mice. Several long-term T cell lines and clones specific for the nonrepetitive Py1 peptide were originated in vitro from both H-2d and H-2b mice. These lines and clones were CD4+ and proliferated in a major histocompatibility complex-restricted fashion. Furthermore, Py1-specific T cell lines and clones did not proliferate in the presence of synthetic peptides from an analogous region of another rodent malaria parasite, P. berghei, despite the high degree of homology existing in this sequence of the two CS proteins. Finally, supernatants from 7 out of 13 clones (from BALB/c mice) produced detectable amounts of interleukin 2 and interferon-gamma; whereas supernatants from the 4 clones from C57BL/6 and 2 from BALB/c mice contained detectable amounts of interleukin 5. These results show that functionally heterogenous CD4+ T cell populations, belonging to either TH1 or TH2 subset, are activated upon immunization of mice with the P. yoelii Py1 synthetic peptide. It is not yet known what differential role these CD4+ subsets play during the malaria infection or after immunization with different malaria T cell epitopes. This knowledge may have a particular impact in the design of effective subunit vaccines against malaria.     

VH and VL region structure of antibodies that recognize the (NANP)3 dodecapeptide sequence in the circumsporozoite protein of Plasmodium falciparum

The sporozoite form of Plasmodium falciparum displays on its surface the circumsporozoite (CS) protein. The central domain of this protein possesses a reiterated tetrapeptide sequence Asn-Ala-Asn-Pro (NANP), and greater than 90% of the sporozoite-specific antibodies obtained from individuals living in malaria endemic areas recognize epitopes within this repeat sequence. Considering the highly repetitive structure of this naturally occurring antigen and its immunodominance, we were interested in analyzing the structural diversity of antibodies that bind to the (NANP)3 sequence. Molecular characterization of immunoglobulin heavy and light chain mRNA was performed for five hybridomas that produce antibodies with binding specificity for the dodecapeptide (NANP)3. These hybridomas were produced in BALB/c mice by inoculation with whole P. falciparum sporozoites. Sequence analysis and Northern blotting showed that for heavy chain, three hybridomas used VH elements that belong to the VHIX family and two to the VHJ558 family. Four different V kappa subgroups were represented among the light chains. Different D and J kappa segments are also utilized, while four heavy chain gene rearrangements involved the JH4 segment. These results indicated that multiple VH-VL gene combinations can code for reactivity to the (NANP)3 sequence, demonstrating that the murine antibody response to this immunodominant region is structurally heterogeneous.     

Induction of humoral immune response against Plasmodium falciparum sporozoites by immunization with a synthetic peptide mimotope whose sequence was derived from screening a filamentous phage epitope library.

The mouse monoclonal antibody 2A10 (immunoglobulin G), which recognizes the (NANP)n repeat of Plasmodium falciparum circumsporozoite surface protein, was used to screen a filamentous phage epitope library expressing random amino acid hexamers. The sequences obtained were TNRNPQ, SNRNPQ, NND-NPQ, SNYNPQ, and QNDNPQ (single-letter amino acid designation). These peptides showed 50% homology with the native epitope (PNANPN) and therefore were considered to mimic its structure (mimotopes). Two of these mimotopes (TNRNPQ and NNDNPQ) inhibited the binding of monoclonal antibody 2A10 to the recombinant protein R32LR, which contains the amino acid sequence [(NANP)15NVDP]2. Immunization of mice and rabbits using the peptide (TNRNPQ)4 induced a humoral response that recognized R32LR by an enzyme-linked immunosorbent assay and P. falciparum sporozoites by an immunofluorescence assay. These results suggest that phage epitope libraries can be exploited to screen for mimotopes in the design of subunit vaccines against infectious agents.     

In vitro immune recognition of synthetic peptides from the Plasmodium falciparum CS protein by individuals naturally exposed to different sporozoite challenge.

The impact of duration and intensity of sporozoite challenge on the in vitro cell immune response to synthetic peptides of the circumsporozoite (CS) protein of Plasmodium falciparum was investigated in residents of a malaria endemic area in Burkina Faso (West Africa). Lymphocyte proliferation and interferon-gamma (IFN-gamma) production were used to assess immune recognition of synthetic peptides corresponding to the polymorphic Th2R and Th3R regions, to the conserved CS.T3 sequence and to NANP and degenerate NVDP repeats. Immune responses were measured in adults and children from a village where they received more than 100 sporozoite inoculations per year and in adults living in a town, exposed to a 10-100 times lower challenge. A lifetime intense exposure apparently increased the ability to proliferate in response to most peptides in the rural adults, who all produced antibodies to NANP repeats. Surprisingly, cell cultures from these subjects seldom contained appreciable levels of IFN-gamma. In the urban adults, possibly due to the moderate challenge they are exposed to, significant differences in the proliferative potentials of the peptides could be detected. The highest stimulation indices were obtained with the genetically unrestricted CS.T3 peptide. Remarkably, proliferative responses to Th2R and Th3R appeared to be correlated with the humoral response to the CS protein, indicating a T helper significance of the epitopes. The differing proliferative potential of the polymorphic epitopes in the urban adults suggests that polymorphism might delay the development of immune responsiveness under conditions of sporadic transmission. The children from the highly malarious village displayed the lowest proliferative scores, accompanied by a high prevalence of antibodies to NANP repeats. On the basis of these findings, the hypothesis is proposed that a pure B cell reactivity to NANP repeats could ontogenetically precede the mounting of a conventional T-B cooperative immune response.     

Immunogenicity of multiple antigen peptides (MAP) containing T and B cell epitopes of the repeat region of the P. falciparum circumsporozoite protein.

The immunogenicity of multiple antigen peptides (MAP) constructs containing T and B cell epitopes of the repeat region of the P. falciparum circumsporozoite (CS) protein was examined in vitro, using a human T cell clone, and in vivo, using four different strains of mice. All the MAP constructs that contained the T cell epitope, (DPNANPNVDPNANPNV), stimulated proliferation and interferon-gamma production by a human T cell clone specific for this epitope which is located in the 5' end of the repeat region of the P. falciparum CS protein. These human T cells did not recognize MAP that contained only the B cell epitope, (NANP)3, which is located in the 3' repeat region. Optimal antibody responses were obtained in mice immunized with MAP containing four copies of tandemly arranged T and B cell epitopes, (TB)4. The murine immune response to the MAP constructs was genetically restricted. Mice of a high responder strain, C57BL, recognized both the 5' and 3' repeat sequences in the MAP as T, as well as B, cell epitopes and developed very high anti-MAP and anti-sporozoite antibody titers. A/J and C3H mice, which were intermediate responders, developed lower antibody titers which varied according to the orientation of the T vs. the B cell epitopes within the MAP constructs. BALB/c mice were nonresponders and did not develop antibodies following immunization with any of the MAP constructs containing the 5' and 3' repeats of the P. falciparum CS protein.     

A linear peptide containing minimal T- and B-cell epitopes of Plasmodium falciparum circumsporozoite protein elicits protection against transgenic sporozoite challenge

An effective malaria vaccine is needed to address the public health tragedy resulting from the high levels of morbidity and mortality caused by Plasmodium parasites. The first protective immune mechanism identified in the irradiated sporozoite vaccine, the "gold standard" for malaria preerythrocytic vaccines, was sporozoite-neutralizing antibody specific for the repeat region of the surface circumsporozoite (CS) protein. Previous phase I studies demonstrated that a branched peptide containing minimal T- and B-cell epitopes of Plasmodium falciparum CS protein elicited antirepeat antibody and CD4(+)-T-cell responses comparable to those observed in volunteers immunized with irradiated P. falciparum sporozoites. The current study compares the immunogenicity of linear versus tetrabranched peptides containing the same minimal T- and B-cell epitopes, T1BT*, comprised of a CS-derived universal Th epitope (T*) synthesized in tandem with the T1 and B repeats of P. falciparum CS protein. A simple 48-mer linear synthetic peptide was found to elicit antisporozoite antibody and gamma interferon-secreting T-cell responses comparable to the more complex tetrabranched peptides in inbred strains of mice. The linear peptide was also immunogenic in outbred nonhuman primates (Aotus nancymaae), eliciting antibody titers equivalent to those induced by tetrabranched peptides. Importantly, the 48-mer linear peptide administered in adjuvants suitable for human use elicited antibody-mediated protection against challenge with rodent malaria transgenic sporozoites expressing P. falciparum CS repeats. These findings support further evaluation of linear peptides as economical, safe, and readily produced malaria vaccines for the one-third of the world's population at risk of malaria infection.     

Synthetic peptides as antigens for the detection of humoral immunity to Plasmodium falciparum sporozoites

The presence of antibodies against P. falciparum sporozoites in humans living in malaria-endemic areas was measured using as antigen the synthetic peptide (NANP)3, which represents the immunodominant region of the circumsporozoite (CS) protein. By using a competitive binding assay it was determined that antibodies which recognize (NANP)3 do not react with a 22-Mer synthetic peptide representing a cross-reacting epitope present in an antigen (5.1) from the blood stages of the parasite. Antibodies present in human sera which react with the 5.1 peptide did not react with (NANP)3. This strongly suggests that antibodies to (NANP)3 found in sera of individuals living in endemic areas are a reflection of exposure to P. falciparum sporozoites. These results validate the use of (NANP)3 for epidemiological studies to detect and measure humoral immunity to P. falciparum sporozoites.     

Multiple T helper cell epitopes of the circumsporozoite protein of Plasmodium berghei

The present findings establish the lack of genetic restriction of the humoral immune response to sporozoites of Plasmodium berghei, corraborating earlier observations that mice of different strains can be protected by immunization with irradiated sporozoites. Most, if not all, anti-sporozoite antibodies are directed against the repetitive B cell epitope of the circumsporozoite (CS) protein. However, neither a peptide containing a dimer of this repeat (17.1), nor a peptide polymer containing multiple repeats induced an antibody response in mice of different H-2 and different genetic backgrounds. A yeast-derived recombinant, containing the repeat domain and part of the surrounding amino and carboxy-terminal regions of the P. berghei CS protein, induces very different levels of antibody in mice of diverse H-2 haplotypes. H-2j mice are high responders and the immunized mice are extensively protected against sporozoite challenge. The lymph node cells of the H-2j mice (but not from other strains) proliferated in the presence of peptide N, contained in the amino terminal region of the CS recombinant. Additional H-2-restricted T cell epitopes have been identified in amino and carboxy-terminal regions of the CS protein, and mice of most of the strains recognized multiple T cell epitopes. Two peptides representing T cell epitopes were synthesized in tandem with a peptide representing the B cell epitope, and were assayed for T helper activity in vivo. The antibody response of mice, primed by a single injection of sporozoites, was boosted very effectively by the administration of peptide N + 17.1 or peptide B-4 + 17.1. The B-4 T cell epitope is located in the carboxy-terminal region of the CS protein and is recognized by mice of at least four different H-2 haplotypes. These observations demonstrate that the immune response to the CS protein of P. berghei is not genetically restricted and that it contains several T cell epitopes, some of which can function as helper epitopes. In addition, they show that a synthetic sporozoite vaccine can boost the immune response to sporozoites.     

Immunogenicity of Well-Characterized Synthetic Plasmodium falciparum Multiple Antigen Peptide Conjugates

Given the emerging difficulties with malaria drug resistance and vector control, as well as the persistent lack of an effective vaccine, new malaria vaccine development strategies are needed. We used a novel methodology to synthesize and fully characterize multiple antigen peptide (MAP) conjugates containing protective epitopes from Plasmodium falciparum and evaluated their immunogenicity in four different strains of mice. A di-epitope MAP (T3-T1) containing two T-cell epitopes of liver stage antigen-1 (LSA-1), a di-epitope MAP containing T-cell epitopes from LSA-1 and from merozoite surface protein-1, and a tri-epitope MAP (T3-CS-T1) containing T3-T1 and a potent B-cell epitope from the circumsporozoite protein central repeat region were tested in this study. Mice of all four strains produced peptide-specific antibodies; however, the magnitude of the humoral response indicated strong genetic restriction between the different strains of mice. Anti-MAP antibodies recognized stage-specific proteins on the malaria parasites in an immunofluorescence assay. In addition, serum from hybrid BALB/cJ x A/J CAF1 mice that had been immunized with the tri-epitope MAP T3-CS-T1 successfully inhibited the malaria sporozoite invasion of hepatoma cells in vitro. Spleen cells from immunized mice also showed a genetically restricted cellular immune response when stimulated with the immunogen in vitro. This study indicates that well-characterized MAPs combining solid-phase synthesis and conjugation chemistries are potent immunogens and that this approach can be utilized for the development of subunit vaccines.     

Novel antigen identification method for discovery of protective malaria antigens by rapid testing of DNA vaccines encoding exons from the parasite genome

We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from the Plasmodium yoelii yoelii genomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protective P. y. yoelii genes can then be identified in the genomic databases of Plasmodium falciparum and Plasmodium vivax and investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192 P. y. yoelii exons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encoding P. y. yoelii circumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs in P. vivax and P. knowlesi but not P. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens.     

Extensive antigenic polymorphism within the repeat sequence of the Plasmodium falciparum merozoite surface protein 1 block 2 is incorporated in a minimal polyvalent immunogen

Polymorphism in pathogen antigens presents a complex challenge for vaccine design. A prime example is the N-terminal block 2 region of the Plasmodium falciparum merozoite surface protein 1 (MSP1), to which allele-specific antibodies have been associated with protection from malaria. In a Zambian population studied here, 49 of 91 alleles sampled were of the K1-like type (the most common of three block 2 types in all African populations), and most of these had unique sequences due to variation in tri- and hexapeptide repetitive motifs. There were significant negative correlations between allelic sequence lengths of different regions of the repeats, so the complete repeat sequence had less length variation than its component parts, suggesting a constraint on overall length. Diverse epitopes recognized by three murine monoclonal antibodies and 24 individual human sera were then mapped by using a comprehensive panel of synthetic peptides, revealing epitopes in all regions of the repeats. To incorporate these different epitopes in a single molecule, a composite sequence of minimal overall length (78 amino acids) was then designed and expressed as a recombinant antigen. More human immune sera reacted with this "K1-like Super Repeat" antigen than with proteins consisting of single natural allelic sequences, and immunization of mice elicited antibodies that recognized a range of five cultured parasite lines with diverse K1-like MSP1 block 2 repeat sequences. Thus, complex allelic polymorphism was deconstructed and a minimal composite polyvalent antigen was engineered, delivering a designed candidate sequence for inclusion in a malaria vaccine.     

Peptide-primed CD4+ cells and malaria sporozoites.

We have mapped a T cell epitope in the circumsporozoite (CS) protein of the murine malaria parasite, Plasmodium yoelii. A 21-mer synthetic peptide corresponding to the amino acid positions 59-79 (referred to as Py1), induced specific proliferation in BALB/c and C57BL/6 mice, and provided help for the production of antibodies to peptides from the repetitive region, (QGPGAP)n, of the same CS protein, when mice were immunized with the Py1 peptide conjugated to the repetitive peptide. Long-term CD3+CD4+CD8-TCR alpha beta+ T cell lines and clones were derived from both strains of mice. These lines and clones, that proliferated in an MHC-restricted fashion, did not recognize peptides from the homologous region of another murine malaria parasite, P. berghei. About 50% of these clones produced detectable amounts of IFN-gamma and IL-2, whereas the remaining produced IL-4, IL-5, and IL-6. In preliminary experiments, some of these clones specifically inhibited P. yoelii sporozoite development in vitro and conferred protection in vivo in passive transfer experiments. These findings show that heterogenous T cell populations are activated in mice upon immunization with a short peptide from the P. yoelii CS protein and that some of these cells could be active in the effector arm of the immune response against malaria sporozoites.     

Antigenic diversity in the circumsporozoite protein of Plasmodium falciparum abrogates cytotoxic-T-cell recognition.

Genetic analysis of field isolates of Plasmodium falciparum has shown selective accumulation of point mutations within the immunologically sensitive sites of the circumsporozoite (CS) protein, a vaccine candidate against malaria. This raised concern whether a vaccine containing the sequence of a selected strain of P. falciparum would be able to confer protection against other variant parasites. The answer to this question remained speculative, and in this study, we have formally tested the immunological impact of such natural variations within a known cytotoxic-T-cell (CTL) epitope, which is recognized by both human and murine CTLs. With a murine model, CTLs were generated against the 7G8 strain of P. falciparum. The ability of these CTLs to lyse histocompatible targets that were pulsed with synthetic peptides corresponding to polymorphic sequences of Brazilian, Papua New Guinean, and The Gambian isolates was determined. While these CTLs were able to recognize three of the four variant CS sequences found in Brazil and Papua New Guinea, they failed to recognize four of the five variant CS sequences found in The Gambia. Among the peptides that lost their reactivity to 7G8-specific CTL, all except one had amino acid variation in more than one residue. On the other hand, only one of the four peptides that showed a positive reaction had amino acid substitutions in more than a single residue. Thus, our findings demonstrate that natural amino acid variations in the CS protein abrogate CTL recognition. Therefore, it is important to consider the implications of these results in designing CS protein-based vaccines.     

T helper epitopes enhance the cytotoxic response of mice immunized with MHC class I-restricted malaria peptides.

We have previously derived MHC class I (H-2Kd) restricted cytotoxic T lymphocytes (CTL) from BALB/c mice immunized with irradiated sporozoites from Plasmodium (P.) berghei and P. yoelii. The CTL recognize synthetic peptides corresponding to a region of the circumsporozoite (CS) protein that is homologous in the two species. In the present study, we have attempted to induce CS-specific CTL by immunization with those peptides in incomplete Freund's adjuvant. Only a low level CTL response was detected in BALB/c mice immunized with synthetic peptides corresponding to the Pb or Py CTL epitopes. In contrast, CS-specific CTL responses could be readily detected in mice injected with mixtures of peptides that combined the P. berghei or P. yoelii CTL epitopes with previously defined T helper epitopes. Several different T helper epitopes were shown to enhance the response when injected as separate peptides in a mixture, or when covalently linked to a CTL epitope. These results may have general implications for the elicitation of CTL responses to defined CTL epitopes and for the design of peptide-based synthetic vaccines.